Clathrin-coated vesicles (ccv) play important roles in sorting plasma membrane proteins into the endocytic pathway and sorting proteins between the trans Golgi network (TGN) and endosomes. These ccv-mediated pathways are fundamental, conserved elements of eukaryotic cells; pathway defects can cause inherited human disorders and are likely to contribute to multigenic diseases such as cancer, heart disease, and Alzheimer's disease. Also, pathogens such as HIV take advantage of these pathways to infect cells and avoid immune surveillance. The overall goal of this project is to understand the molecular basis of selective protein transport by ccv in normal cells to provide a foundation for understanding how defects can lead to disease. Towards this goal, ccv-mediated protein transport has been characterized in the yeast Saccharomyces cerevisiae. In earlier studies we characterized a network of clathrin adaptors that function in traffic between the TGN and endosomes. Gga proteins and AP-1 constitute major hubs in this network. During the previous funding period we discovered that Gga proteins and AP-1 are recruited sequentially to the TGN to form distinct clathrin coats. This process of adaptor progression is regulated by levels of the phosphoinositide PI4P that are generated by the TGN PI4-kinase Pik1p, which appears to be recruited to the TGN by direct interaction with Gga proteins. Based on these findings we have proposed a model in which a positive feedback loop between Gga proteins and Pik1p regulates progressive assembly of adaptor-enriched ccv at the TGN. Our studies of adaptor progression reveal previously unappreciated principles for regulation of ccv formation and offer a novel paradigm for assembly of functionally-specialized coated vesicles at an organelle. Thus, our findings have opened up unique avenues to address fundamental aspects of eukaryotic membrane traffic. A combination of genetic, biochemical, and live cell imaging strategies will be applied to achieve three specific aims. First, we will apply live cell imaging and genetic strategies to functionally define the process of sequential adaptor- specific ccv formation and relate it to other vesicle trafficking pathways emanating from the TGN. Second, complementary biochemical approaches will be used to characterize the molecular basis for regulation of adaptor progression with an emphasis on testing the Gga-Pik1p positive feedback model and defining adaptor interactions with PI4P. Third, approaches in the first two aims will be extended to assess the functions of conserved TGN/endosome accessory factors in ccv formation. Together these studies are expected to provide significant insights into the fundamental process of ccv formation in pathways between the TGN and endosomes, thereby helping to establish a foundation for understanding the roles these processes play in human disease.